A connectivity-based synaptome

描述（由申请人提供）：突触病理是各种神经退行性和神经发育性疾病的常见病理。对于许多这样的疾病，包括精神分裂症和自闭症谱系障碍，突触组成的变化被怀疑与一些最具破坏性的症状有因果关系。通过电子显微镜观察，大脑中的突触共享关键的结构蛋白，这可以从它们相似的、易于识别的结构中得到证明，但它们也非常不均匀：只有少数突触成分是所有突触共享的。突触前膜粘附突触后膜在一个特别强的相互作用，抵抗解体。这种相互作用可以抵抗组织分离，并且在过去的几十年里已经被用于纯化突触体：突触前到突触后的粘附和附着的膜重新密封以形成封闭的突触细胞器。目前的蛋白质组学研究已经利用突触体或这些制剂的亚组分来评估突触的组成。虽然这支持了使用突触体进行基于蛋白质组学的比较的可行性，但由此产生的大型数据集并不是很有用，因为它们反映了一个高度复杂的起始材料，包含了巨大的异质突触群体。突触的异质性很大程度上是基于连接的差异，但目前还没有可用的方法可以用来询问和分析已识别的突触群体之间的差异。在这里，我们提出了一种基于哺乳动物跨突触伙伴GFP重构技术的新颖而直接的方法，该方法将能够在健康和疾病中确定的突触种群之间进行蛋白质组学比较。我们已经组建了一个团队，用定义明确的突触电路来测试这一点。这种方法将是向前迈出的重要一步，因为它将允许对可以从复杂电路中分离出来的特定突触种群进行快速、大规模的评估。

DESCRIPTION (provided by applicant): Synapse pathology is common to a variety of neurodegenerative and neurodevelopmental diseases. For many such disorders, including schizophrenia and autism spectrum disorders, changes in synapse composition are suspected to be causally related to some of the most devastating symptoms. Synapses throughout the brain share key structural proteins evidenced by their similar, readily identified structure as see through an electron microscope, but they are also very heterogeneous: only a handful of synapse components is known to be shared by all synapses. Presynaptic membranes adhere to postsynaptic membranes in an exceptionally strong interaction, resistant to disassembly. This interaction can withstand tissue fractionation and has been exploited over the past several decades to purify synaptosomes: pre- to postsynaptic adhesions and attached membranes that re-seal to make an enclosed synaptic organelle. Current proteomics studies have utilized synaptosomes or subfractions of such preparations to assess synapse composition. While this supports the feasibility of using synaptosomes for proteomics-based comparisons, the resulting large datasets have not been very useful since they reflect a highly complex starting material containing an enormously heterogeneous population of synapses. Synapse heterogeneity is based largely on differences in connectivity, but currently there are no methodologies available that can be used to interrogate and analyze differences between identified synapse populations. Here we propose a novel and straightforward method based on the mammalian GFP reconstitution across synaptic partners technique that will enable proteomic comparisons between identified synapse populations in health and disease. We have assembled a team to test this using well- defined synaptic circuits. This approach will be a major step forward as it wll permit a rapid, large-scale assessment of particular synapse populations that can be isolated from complex circuits. PUBLIC HEALTH RELEVANCE: Synapse pathology is common to a variety of neurodegenerative and neurodevelopmental diseases. The proposed studies will develop methodologies designed to identify the entire set of proteins, the proteome, expressed at the synapses of an identified circuit. This will enable future studies in which changes in protein composition at specified synapses can be evaluated in mouse models relevant to human brain disorders, an important step toward identifying relevant targets for therapy.